The long range goal of this research is to elucidate the molecular basis for cytoskeletal control of the membrane and organelle movements characteristic of eukaryotic cell plasma membranes. This proposal will focus on the dynamic organization of the human erythrocyte membrane cytoskeleton and its role in the modulation of erythrocyte shape and deformability. Recently, I have identified a non-muscle form of tropomyosin in human erythrocytes that is present in sufficient quantities to almost completely coat all of the short actin filaments in the membrane cytoskeleton. The functional implications of tropomyosin for the organization of the spectrin-actin cytoskeletal network and the control of cell shape will be investigated as follows. (1) The effect of tropomyosin on spectrin binding to F-actin will be examined, in the presence and absence of band 4.1, using quantitative F-actin binding assays with radiolabeled proteins, in order to determine whether both spectrin and tropomyosin could be bound to the same regions of the short actin filaments thought to exist in the membrane-cytoskeleton. (2) Association of tropomyosin with the cytoskeleton in situ will be examined by immunoblotting analysis of cytoskeletons isolated by non-ionic detergent extraction of intact cells and ghosts and by immunofluorescence localization in 0.5Mum frozen sections of intact cells and ghosts. The effect of micromolar calcium-induced discocyte-echinocyte shape transformations on the association of tropomyosin with the membrane-cytoskeleton will be explored. (3) Cytoskeletal control of calcium and MgATP-dependent discocyte-echinocyte shape transformations will be studied in a lysed cell system. Selective extraction of tropomyosin and other loosely bound components from ghosts will be correlated with the ability of ghosts to change shape. Restoration of regulated shape changes in extracted ghosts by reconstitution with purified components will permit direct evaluation of the functional significance of tropomyosin or other potential regulatory components (e.g., calmodulin, kinases, phosphatases). (4) The existence of a true-myosin-like enzyme in erythrocytes will be reinvestigated, as will functional homologies of tropomyosin-binding proteins to muscle troponins. This might mean that tropomyosin could be a regulatory component in an erythrocyte contractile apparatus responsible for erythrocyte shape transformations.